*3.1. Clinical Outcomes*

After six months of therapy, no patient discontinued adalimumab due to adverse effects and 100% of the patients achieved clinical remission, but the success of the therapy was only achieved in 65% of patients (13 out of 20), namely the remaining seven on corticosteroid therapy. CRP decreased from a median value of 6.5 mg/<sup>L</sup> (0.7–45.5 mg/L) at T0 to a median value of 2.9 mg/<sup>L</sup> (0.1–16.5 mg/L) at T1 (*p* = 0.010). Similarly, erythrocyte sedimentation rate (ESR) decreased from the median value of 22 mm/h (1–94 mm/h) at T0 to 9 mm/h (4–60 mm/h) at T1 (*p* = 0.020). Calprotectin decreased from a median value of 262 ug/g (35–726 ug/g) at T0 to a median value of 80 ug/g (39–969 ug/g) at T1 (*p* = 0.035) (Figure 1).

**Figure 1.** Serum and faecal inflammatory biomarkers trend after six months of adalimumab therapy.

*3.2. Trend of Microbiota During Therapy*

Focusing on the temporal trend, regarding the phyla, *Firmicutes* rose from 45.5 ± 5.1% at T0 to 48.9 ± 3.0% at T1 (*p* = 0.523), *Bacteroidetes* from 33.5 ± 4.7% at T0 to 37.1 ± 4.0% at T1 (*p* = 0.411), *Proteobacteria* fell from 15.7% ± 3.5% at T0 to 10.3 ± 3.4% at T1 (*p* = 0.038). Finally, the *Actinobacteria* increased from 2.6% ± 0.7% at T0 to 3.0% ± 0.7% at T1 (*p* = 0.928) (Figure 2).

**Figure 2.** Per cent composition of phyla of bacterial microbiome at baseline and six months after starting adalimumab therapy.

Regarding the bacterial families, that of *Lachnospiraceae* was the most represented both at T0 (18.2 ± 2.6%), and at T1 (23.6 ± 2.2%), without statistical difference between these two periods (*p* = 0.100). Regarding the species, *Ruminococcus gnavus* decreased from 3.3 ± 1.8% at T0 to 1.6 ± 0.3% at T1 (*p* = 0.350); *Bacteroides ovatus* rose from 2.9 ± 0.9% to 2.4 ± 0.6% (*p* = 0.540); *Faecalibacterium prausnitzii* rose from 3.7 ± 1.2% to 2.2 ± 0.8% (*p* = 0.130), *Bifidobacterium adolescentis* decreased from 1.3 ± 0.5% to 1.2 ± 0.5% (*p* = 0.260); *Escherichia coli* did not change (11.4%, *p* = 0.998).

Baseline microbiota changes in relation to success or therapeutic failure are reported in Table 3.



We also analysed the trend in composition of microbiome in relation to therapeutic success. Regarding bacterial phyla, Proteobacteria decreased in patients in whom therapeutic success was obtained, passing from a value of 15.8 ± 4.6% to 6.8 ± 3.1% (*p* = 0.049), while in non-responders, their percentage did not change (T0 = 15.6 ± 5.7% *vs.* T1 = 16.8 ± 7.6%, *p* = 0.890). The data regarding changes in composition of the microbiome in responders and in non-responders to adalimumab therapy are shown in Table 4.

**Table 4.** Trend in phyla, family and bacterial species according to pharmacological success or failure.


In Table 5, the microbiome trend is reported according to CRP values after six months of adalimumab therapy.

With regards to the Lachnospiraceae family in patients with normalization of CRP levels after six months of adalimumab therapy, at T0 it showed a mean value of 16.6 ± 3.1% and at T1 this increased to 23.9 ± 2.6% among bacterial families (*p* = 0.049).

According to disease localization, the phylum Actinobacteria was more represented if the colon was inflamed (3.9 ± 1.0%) compared to an ileal CD (0.7 ± 0.5%); the differences among the other phyla were not statistically significant (Table S1). The changes in phyla according to disease localization were not statistically significant (Table S2).


**Table 5.** Trend of the intestinal microbiome between T0 and T1 according to C-reactive protein levels at six months.

According to disease severity, the phylum Bacteroidetes was much more represented in patients with mild or moderate endoscopic activity (41.4 ± 4.5%), compared to patients with severe endoscopic activity (15.0 ± 7.5%) (*p* = 0.006); the phylum Proteobacteria was more represented in patients with severe endoscopic activity (25.2 ± 8.6%) compared to patients with mild or moderate endoscopic activity (11.7 ± 3.0%) (*p* = 0.076) (Table S3). The changes in phyla according to endoscopic disease activity were not statistically significant (Table S4).
